Formation of Microscale Gradients of Protein Using Heterobifunctional Photolinkers

Abstract

Gradients of biological molecules on a microscale have been postulated to elicit cellular responses, such as migration. However, it has been difficult to prepare such gradients for experimental testing. A means for producing such gradients has been developed using a heterobifunctional photolinking agent with laser light activation. The photolinking agent synthesized includes an N-hydroxysuccinimide group and a photoreactive benzophenone (BP) separated by a tetraethylene glycol (TEG) spacer. The presence of the tetraethylene glycol spacer renders the photolinker hydrophilic, a desirable trait for conjugation in aqueous solutions. The linker was then conjugated to R-phycoerythrin (R-PE), a fluorescent protein. The resulting photolinker-R-phycoerythrin conjugate (BP-TEG-PE) was then immobilized onto a polystyrene surface by laser irradiation on a motorized stage. By varying exposure time of the sample to the beam, the amount of BP-TEG-PE immobilized on the surface was changed over an order of magnitude over a distance of 250 microns. This method can be applied to prepare gradients of proteins that elicit biological responses, such as extracellular matrix proteins or growth factors, and to study the biological effects of such gradients.

Defining the concentration gradient of nerve growth factor for guided neurite outgrowth

“…Diffusible and contact-mediated cues are probably important to CNS regeneration and a hydrogel matrix loaded with such cues may provide a vital addition to the current device designs 5,23,42 that are intended to augment CNS nerve regeneration. However, there are signi®cant challenges to overcome prior to clinical application.…”

“…If the source and the sink concentrations are not maintained, the gradient will quickly diminish within 48 h. To translate this model to a device, we are developing an immobilized concentration gradient of neurotrophic factors in a threedimensional hydrogel which may overcome challenges associated with the unstable concentration gradient discussed above. This well-de®ned diffusible cue-loaded hydrogel, in conjunction with contact-mediated cues, 5,23,42 may open a new horizon in the quest to enhance axonal regeneration after injury. where C gi is the concentration of solute ªiº on the gel side of the donor solution±gel interface, L is the thickness of the membrane and D i is the diffusion coef®cient of substance ªiº.…”

Surface modification of titanium, titanium alloys, and related materials for biomedical applications

“…Weber et al [183] grafted carbohydrates onto TiO 2 surfaces for applications in cell recognition, blood coagulation cascade and sialoglycoprotein recognition. Hypolite et al [184] and Herbert et al [185] synthesized bioconjugate molecules bearing both a peptide domain and photoactive group together with benzophenone. The benzophenone derivatives could be linked covalently onto the solid surface by irradiation.…”

Poly(l-lysine)-grafted-poly(ethylene glycol)-based surface-chemical gradients. Preparation, characterization, and first applications

“…53,54 Some of these surfaces have been used to study cell growth. Other techniques have been used to gradually immobilize biomolecules, such as proteins, on surfaces, for example, microfluidics, 55-57 covalent coupling to an alkanethiol gradient, 58,59 covalent coupling by laser irradiation, 60 the use of stamping techniques and electrophoretics, 61 ink-jet printing, 62,63 drainage, 64 or by controlling the adsorption kinetics. 65 In this study we present the generation and characterization of PLL-g-PEG gradients prepared by means of an immersion process originally developed for alkanethiols ͓Fig.…”